Control of cotton boll weevil, alfalfa weevil, and corn rootworm via contact with a strain of Bacillus thuringiensis

ABSTRACT

The subject invention concerns a novel and useful insecticide with activity against insect pests of cotton, potato, alfalfa and corn crops. These pests do heavy damage to the crops. The insecticide of the subject invention is a B. thuringiensis microbe given the designation strain san diego. The spores or crystals of this microbe are useful to control the cotton boll weevil, the Colorado potato beetle, the alfalfa weevil, and the corn rootworm.

This is a continuation-in-part application of our copending applicationU.S. Ser. No. 714,790, filed Mar. 22, 1985, now U.S. Pat. No. 4,764,372.

DESCRIPTION Background of the Invention

Bacillus thuringiensis (Bt) produces an insect toxin designated asδ-endotoxin. It is synthesized by the Bt sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insectlarvae, is transformed into biologically active moieties by the insectgut juice proteases. The primary target is insect cells of the gutepithelium, which are rapidly destroyed.

The reported activity spectrum of Bt covers insect species within theorder Lepidoptera, many of which are major pests in agriculture andforestry. The activity spectrum also includes the insect order Diptera,which includes mosquitoes and black flies. See Couch, T. L., (1980)"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"Developments in Industrial Microbiology 22 :61-76 Beegle, C. C., (1978)"Use of Entomogenous Bacteria in Agroecosystems," Developments inIndustrial Microbiology 20:97-104. Krieg, et al., Z. ang. Ent. (1983)96:500-508, describe a Bt isolate named Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are Colorado potato beetle, Leptinotarsa decemlineata,and Agelastica aini.

In our parent application, Ser. No. 714,790, we disclose a novel Btisolate active against Coleoptera. There is no disclosure of activityagainst the cotton boll weevil (Antnonomus grandis), tne Colorado potatobeetle (Leptinotarsa decemlineata), the alfalfa weevil (H-ypera postica)or the corn rootworm (Diabrotica longicornis). We have also subsequentlydiscovered that this Bt isolate is not active against all Coleopteratested. See Table 1, infra.

COTTON BOLL WEEVIL

The cotton boll weevil is easily one of the most notorious agriculturalpests in the world. It occurs in all the principal cotton growing areasof Central and South America and the United States except parts ofCalifornia. Wherever it is presant it is the key pest in cotton. Theinjury is caused by the adults and the larvae or grubs. The adultweevils chew into or puncture the squares and bolls, and with their longslender bills, feed on the inner tissues. The eggs are laid in theseholes and the hatching grubs bore into the boll or square, causing thesquares to drop off or wither and dry on the plant. This feeding eitherresults in direct destruction of the flower or reduction of fibercontent in the boll. Losses can be so great as to be limiting. In 1982damage to cotton in the U.S. was estimated at $429 million. This figureis expected to continue to increase.

Chemical insecticides and cultural controls are currently empoyed in thecontrol of boll weevil. These have associated problems and are notcompletely effective. There is a definite need for alternative materialsthat could be used in a complementary fashion with existing controls andto replace control agents that may lose efficacy due to resistance orother factors.

COLORADO POTATO BEETLE

The Colorado potato beetle (CPB) is the most important defoliatinginsect pest of potatoes worldwide and is of particular importance in thenortheastern United States, Europe and the Soviet Union. In the U.S. itis a key insect pest of potatoes in all the majrr potato-producing areasof the U.S. except California. This is an area representing 750,000acres of potatoes. This species is also a serious pest on eggplant,tomato, and pepper.

Both the adults and larvae feed on the foliage. In most areas, two tothree generations occur each year. If left unchecked this pest canseverely defoliate a crop, often consuming all of the above-groundportions of the host plant.

Chemical pesticides are used to control this pest: however, one of theprincipal problems in the use of this strategy is the ability of thebeetle to rapidly develop resistance to new chemical insecticides.Generally 2 to 10 applications of insecticide are made to control CPB,depending on the levels of insecticide resistance, population densityand number of generations per year. Resistance to virtually allavailable insecticides has been documented in some areas, notably LongIsland, N.Y. This has made it practically impossible to grow potatoes inthese areas. All of this indicates the urgency of finding new agents forthe control of CPB, particularly agents that have a mode of action verydifferent from the conventional insecticides currently being used. TheBt strain described here is such an agent. It acts as a proteinaceousstomach poison as opposed to a contact nerve poison. The importance ofCPB and the urgency of finding insecticides to which this pest is notresistant make it imperative that new insecticidal agents like thestrain described herein be developed as rapidly as possible.

ALFALFA WEEVIL

The alfalfa weevil, Hypera postica, and the closely related Egyptianalfalfa weevil, Hyoera brunneipennis, are the most important insectpests of alfalfa grown in the United States,with 2.9 million acresinfested in 1984. An annual sum of 15 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

The larval stage is the most damaging in the weevil life cycle. Byfeeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

Chemical insecticides play a major role in effective alfalfa weevilcontrol. However, there are several problems associated with their useincluding:

1. acute mammalian toxicity: several of the most effective insecticidesused for weevil control are highly toxic to humans and other mammals,and are sold on a restricted basis in many states. Toxic residues are anadditional problem for hay sold as feed for livestock.

2. honeybee toxicity: the honeybee is sensitive to some of theinsecticides used for alfalfa weevil control. Because alfalfa is themajor source of nectar for commercial honeybee colonies in the U.S., theuse of insecticides with honeybee toxicity is incompatible with theneeds of the honey producers.

3. toxicity to natural enemies: the insect parasites and predators whichnormally help control populations of minor alfalfa pests (aphids,leafhoppers, caterpillars) are highly susceptible to all insecticidespresently used for alfalfa weevil control. Reductions in the numbers ofbeneficial insects can result in increased populations of these onceminor pests, and in the consequent application of additionalinsecticides.

CORN ROOTWORM

Approximately 9.3 million acres of U.S. corn is infested with the cornrootworm species complex, which includes the northern corn rootworm,Diabrotica longicornis, the southern corn rootworm, D. undecimpunctata,and the western corn rootworm, D. virgifera. The soil-dwelling larvae ofthese Diabrotica species feed on corn root, causing lodging of the cornplant. This eventually results in yield reduction or death of the plant.By feeding on cornsilks the adults reduce pollination and, therefore,the yield of corn per plant. In addition, adults and larvae of thesouthern corn rootworm, also known as the spotted cucumber beetle,attack cucurbit crops (cucumbers, squash, melons, etc.) and manyvegetable and field crops in commercial production, as well as in homegardens.

Control of the corn rootworm has been partially addressed by culturalmethods, such as crop rotation and application of high nitrogen levelsto stimulate the growth of adventitious root systems. However, chemicalinsecticides are relied upon heavily to guarantee the desired level ofcontrol. Insecticides are banded onto the soil or incorporated into thesoil. The major problem associated with the use of these chemicals isthe development of resistance among the treated insect populations.

BRIEF SUMMARY OF THE INVENTION

Disclosed and claimed is the use of our Bacillus thuringiensis isolateto control the cotton boll weevil (Anthonomus grandis), the Coloradopotato beetle (Leptinotarsa decemlineata), the alfalfa weevil (Hyperapostica) and the corn rootworm (Diabrotica longicornis). This B.thuringiensis isolate, initially designated "M-7", has now been named B.thuringiensis strain san diego. (B.t.sd).

Because the boll weevil and the Colorado potato beetle are notiniigenous to California, we have developed a bioassay using the elmleaf beetle (Pyrrhalta luteola). Both adults and larvae of this speciesshow greater than 90% mortality when exposed to host plant leavestreated with B.t.sd spore/crystal preparations at 10⁴ spore equivalentsper square centimeter of leaf surface. In contrast, treatment of theseinsects with the lepidopteran-specific strain of Bacillus thuringiensisHD-1 at 10⁶ spore equivalents/cm² gave less than 5% mortality.

B.t.sd has shown significant levels of activity against larvae of theEgyptian alfalfa weevil and the southern corn rcotworm.

DETAILED DISCLOSURE OF THE INVENTION

The Bacillus thuringiensis isolate used in the subject invention,designated B.t.sd, is unusual in having a unique parasporal body(crystal) which under phase contrast microscopy is dark in appearancewith a flat, square configuration.

A subculture of B. thuringiensis strain san diego has been deposited inthe permanent collection of the Northern Regional Research Laboratory,U.S. Department of Agriculture, Peoria, Ill., U.S.A. on Feb. 27, 1985.The culture was assigned the accession number NRRL B-15939 by therepository. This deposit is available to the public upon the grant of apatent disclosing it. The deposit is also available as required byforeign patent laws in countries wherein counterparts of the subjectapplication, or its progeny, are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action. B. thuringiensis strain san diego, NRRL B-15939,can be cultured using standard art- media and fermentation techniques.Upon completion of the fermentation cycle the bacteria can be harvestedby first separating the Bt spores and crystals from the fermentationbroth by means well known in the art. The recovered Bt spores andcrystals can be formulated into a wettable powder, liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. These formulation andapplication procedures are all well known in the art and are used withcommercial strains of B. thuringiensis (HD-1) active againstLepidoptera, e.g., caterpillars.

Formulated products can be sprayed or applied onto foliage to controlphytophagous beetles, or formulated bait granules containing anattractant and spores and crystals of B.t.sd can be applied to the soil.Formulated B.t.sd can also be applied as a seed-coating or roottreatment or total plant treatment at late stages of the crop cycle.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE l Culturing B. thuringiensis strain san diego NRRL B-15939.

A subculture of B. thuringiensis strain san diego NRRL B-15939 can beused-to inoculate the following

    ______________________________________                                        Tryptone             10     gm                                                Yeast extract        5      gm                                                NaCl                 5      gm                                                5 N NaOH             0.6    ml                                                Water                1000   ml                                                ______________________________________                                    

As per standard microbiological techniques, the above medium would besterilized prior to inoculation and the inoculation would be done usingaseptic procedures.

A procedure that has produced good results is as follows:

A series of 150 ml Erlenmeyer flasks containing sterila PWYE medium(peptone 5.0%; yeast extract 0.1%: NaCl 0.5% in 1 liter of water: adjustpH to 7.5) are inoculated from a petri plate culture of B. thuringiensisM-7, NRRL B-15939. The flasks are incubated at 30° C. on a rotary shaker(200 rpm) overnight. From this starter culture, 300 ml of LB broth in a2 liter flask is inoculated using 7.5 ml of the starter. The LB-brothflasks are incubated under the same conditions as the stater, but areharvested after 4 days.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The Bt spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Testing of B. thuringiensis strain san diego NRRL B-1593Sporesand Crystals

B. thuringiensis strain san diego NRRL B-15939 spores andcrystals,obtained as described above, were tested against variousinsects by use of the following procedures: Assays against Diabroticaundecimpunctata undecimpunctata, Leptinotarsa decemlineata and Pyrrhaltaluteola were carried out by spraying spore/crystal preparations, orpurified crystals,onto leaf discs from appropriate host plants. Assaysagainst Aedes aegypti were performed by adding spore/crystalpreparations to larvae in water. All other assays were carried out byincorporating spore/crystal preparations, or purified crystals, into anappropriate standard diet mixture. More specifically, the assay for theColorado potato beetle and the boll weevil were conducted as follows:

A. B.t.sd spore/crystal preparations of varying concentrations wereprepared as described in Example 1. Potato leaves were dipped in thesesolutions, air dried for 1 hr and exposed to 2nd instar Colorado potatobeetle larvae. Each dosage was tested against 20 larvae and repeated 4times. Mortality was determined after 48 hr. The LC-50 was determined byprobit analysis. This procedure yielded an LC-50 of 7.6×10⁶ spores/ml ofsolution. LC-50 referes to a lethal concentration that kills 50% oflarvae.

B.t.sd spore/crystal preparations of varying concentrations wereprepared as described in Example 1, and incorporated into a standardboll weevil diet. Second instar boll weevil larvae were introduced tothe diet and mortality was assessed after 48 hr. Each dosage of B.t.sdagainst 20 larvae and repeated 4 times. The LC-50 was determined byprobit analysis (Finney, D. J. 1971 Probit Analysis 3d ed. CambridgeUniversity Press, Cambridge). This procedure gave an LC-50 of 4.65×10⁵spores/ml of diet.

EXAMPLE 3 Activity of B.t.sd Against Alfalfa Weevil

B.t.sd spore/crystal preparations of varying concentrations wereprepared as described in Example 1. Sprigs of alfalfa were dipped inthese solutions and air dried for 15 min. Each sprig was then put into asmall vial of water, alfalfa weevil larvae were added to the leaves, andthe entire assemblage placed in an enclosed rearing chamber. Each dosagewas tested against 20 2nd instar larvae and replicated 3 times. After 96hr, the highest dosage tested, 7.6×10⁷ spores/ml solution, caused 80%mortality. The surviving weevil larvae were stunted and were notfeeding. Lower concentrations resulted in only minimal levels ofmortality, but caused significant levels of feeding inhibition. It islikely that these feeding-inhibited larvae will not survive to adulthoodin the field.

EXAMPLE 4 Activity of B.t.sd Against Southern Corn Rootworm

B.t.sd spore/crystal preparations of varying concentrations wereprepared as described in Example 1. Germinated corn kernels were dippedinto the solutions and air dried for 15 min. Each kernel was placed in apetri dish which contained a thin layer of sterila moist sand, and 102nd instar corn rootworm larvae were placed on each kernel. Eachconcentration was tested against 20 2nd instar larvae and replicated 3times. After 96 hr 50% of the larvae treated at the highest dosage(1×10¹⁰) spores/ml of solution) were dead and the survivors were nolonger feeding.

                                      TABLE 1                                     __________________________________________________________________________    Insects Evaluated for Susceptibility to Bacillus thuringiensis strain san     diego                                                                         ORDER  FAMILY  SPECIES  COMMON NAME STAGES TESTED                                                                           ACTIVITY                        __________________________________________________________________________    Coleoptera                                                                           Chrysomelidae                                                                         Diabrotica                                                                             Western spotted                                                                           A         -                                              undecimpunctata                                                                        cucumber beetle                                                                           L         ++                                             undecimpunctata                                                               Haltica tom-         A,L       +++                                            bacina                                                                        Leptinotarsa                                                                           Colorado potato                                                                           L         +++                                            decemlineata                                                                           beetle                                                               Pyrrhalta                                                                              Elm leaf beetle                                                                           A,L       ++++                                           luteola                                                               Curculionidae                                                                         Anthonomus                                                                             Boll weevil A         +++                                            grandis              L         ++++                                           Otiorhynchus                                                                           Black vine weevil                                                                         L         ++                                             sulcatus                                                                      Hypera   Egyptian alfalfa                                                                          L         +++                                            brunneipennis                                                                          weevil                                                       Dermestidae                                                                           Attagenus                                                                              Black carpet beetle                                                                       L         -                                              megatoma                                                              Ptinidae                                                                              Gibbium  --          A         -                                              psylloides                                                            Tenebrionidae                                                                         Tenebrio Yellow mealworm                                                                           L         ++                                             molitor                                                                       Tribolium                                                                              Red flour beetle                                                                          A,L       -                                              castaneum                                                      Diptera                                                                              Culicidae                                                                             Aedes aegypti                                                                          Yellow fever mosquito                                                                     L         -                               Lepidoptera                                                                          Noctuidae                                                                             Spodoptera                                                                             Beet armyworm                                                                             L         -                                              exigua                                                                        Trichoplusia                                                                           Cabbage looper                                                                            L         -                                              ni                                                             __________________________________________________________________________     Assays against Diabrotica undecimpunctata undecimpunctata, Leptinotarsa       decemlineata and Pyrrhalta luteola were carried out by spraying               spore/crystal preparations, or purified crystals, onto leaf discs from        appropriate host plants. Assays against Aedes aegypti were carried out by     adding spore/crystal preparations to larvae in water. All other assays        were carried out by incorporating spore/crystal preparations or purified      crystals into an appropriate diet mixture. Insecticidal activity was          arbitrarily classified from weak (+) to very strong (++++). A: adults, L:     larvae.                                                                  

We claim:
 1. A process, for controlling cotton boll weevil whichcomprises contacting the host plant of said cotton boll weevil with acotton boll weevil-controlling effective amount of Bacillusthuringiensis strain san diego, having the identifying characteristicsof NRRL B-15939, or toxic crystals or spores from said strain.
 2. Aprocess, according to claim 1, which comprises incorporating saidBacillus thuringiensis strain san diego, or toxic crystals orspores™from said strain, into a bait granule and placing said granule onor in the soil when planting seeds of cotton, or at later stages of thecrop cycle.
 3. A process for controlling alfalfa weevil which comprisescontacting the host plant of said alfalfa weevil with an alfalfaweevil-controlling effective amount of Bacillus thuringiensis strain sandiego, having the identifying characteristics o.f NRRL B-15939, or toxiccrystals or spores from said strain.
 4. A process, according to claim 3,which comprises incorporating said Bacillus thuringiensis strain sandiego, or toxic crystals or spores from said strain, into a bait granuleand placing said granule on or in the soil when planting seeds ofalfalfa, or at later stages of the crop cycle .
 5. A process forcontrolling corn rootworm which comprises incorporating in the soilhabitat of said corn rootworm a corn rootworm-controlling effectiveamount of Bacillus thuringiensis strain san diego, having theidentifying characteristics of NRRL B-15939, or toxic crystals or sporesfrom said strain.
 6. A process, according to claim 5, which comprisesincorporating said Bacillus thuringiensis strain san diego, or toxiccrystals or spores from said strain, into a bait granule and placingsaid granule on or in the soil when planting seeds of corn, or at laterstages of the crop cycle.